Human tissue-type plasminogen activator converts plasminogen to plasmin. The plasmin, so produced, proteolytically cleaves fibrin matrices which comprise the backbone of blood clots. Human tissue-type plasminogen activator thereby mediates the dissolution of blood clots and is consequently useful in the treatment of various thrombotic disorders.
The abbreviation "t-PA" for human tissue-type plasminogen activator was adopted after proposal at the XXVIII Meeting of the International Committee on Thrombosis and Hemostatis, Bergamo, Italy, July 27, 1982. As used herein, the terms "human tissue-type plasminogen activator", "human t-PA", "t-PA", "human tissue plasminogen activator" or "tissue plasminogen activator" denote human extrinsic (tissue-type) plasminogen activator, produced, for example, from natural source extraction and purification [see Collen et al., European Patent Application No. 41766 (published Dec. 16, 1981 based upon a first filing of June 11, 1980) and Rijken et al., Journal of Biol. Chem. 256, 7035 (1981), incorporated herein by reference], and by recombinant cell culture systems as described together with the amino acid sequence and other characteristics of the molecule, for example, in European Patent Application Publication No. 93619, (published Nov. 9, 1983 based upon a first filing of May 5, 1982), incorporated herein by reference. The terms also cover biologically active human tissue-type plasminogen activator equivalents, differing in glycosylation patterns, which are thought to be dependent on the specific culture conditions used and the nature of the host from which the human tissue-type plasminogen activator is obtained, and differing in one or more amino acid(s) in the overall sequence.
Researchers in Assignee's laboratories produced human tissue-type plasminogen activator, essentially free of proteins with which it is ordinarily associated, via recombinant DNA technology in prokaryotic and eukaryotic hosts. (See EPA 93619, supra.) Several reasons attend the preferable production of human tissue-type plasminogen activator in recombinant eukaryotic hosts, such as mammalian cells. Eukaryotic hosts in general are efficient in their ability to recognize and glycosylate specific amino acid residues in the human tissue-type plasminogen activator molecule which are ordinarily glycosylated in the native state, generate the naturally occurring covalent cross-linkages between various cysteine residues of the human tissue-type plasminogen activator molecule, and more closely approximate the overall conformational structure of native human tissue-type plasminogen activator. These features are thought to be important for producing a biologically active and safe human tissue-type plasminogen activator product.
However, the production of human tissue-type plasminogen activator from recombinant host cells is not without attendant problems. Thus, it has been found that yield-limiting difficulties arise because the cells producing human tissue-type plasminogen activator are customarily cultured in the presence of serum, various fractions derived from blood or other animal tissues or hydrolysates thereof. As a consequence, the human tissue-type plasminogen activator secreted by such cells is exposed to large amounts of serum proteins and other serum components. It was found that certain of these components invariably complicate the purification of an intact human tissue-type plasminogen activator to a pharmaceutically acceptable form because they form difficult-to-separate bound aggregate complexes with the human tissue-type plasminogen activator molecule. These aggregates also interfere with the biological activity of the human tissue-type plasminogen activator molecule, thus reducing overall the ordinarily expected yields of biologically active intact human tissue-type plasminogen activator. Addition of serum components also increases the mass of impurities that must be removed during purification. Further, many of these components proteolytically degrade tissue-type plasminogen activator. Purification of the desired human tissue-type plasminogen activator from these systems pose technical difficulties, consequently requiring more expensive procedures.
Consistent with these observations, serum-free media have been suggested in attempts to avoid problems in producing and purifying endogenously produced plasminogen activators secreted by cultured cells. See, for example, 1) European Patent Application Publication No. 113319 (published July 11, 1984, based upon a first filing of December 30, 1982) which discloses the preparation of serum-independent natural human cell lines secreting endogenously-produced human tissue-type plasminogen activator, 2) U.S. Pat. No. 4,232,124, 3) U.S. Pat. No. 4,328,314, 4) U.S. Pat. No. 4,317,882, and 5) Gasser et al., In Vitro Cellular and Developemental Biology 21, 588 (1985). None of these references relate to high density cell growth, and in particular to recombinant suspension host cell cultures. On the other hand, European Patent Application Publication No. 112940 (published July 11, 1984, based upon a first filing of Dec. 30, 1982) is directed to a process of producing human tissue-type plasminogen activator involving the addition of albumin, a protein component of serum.
Various means for fractionating cell cultures to remove, for example, serum components are also known. See, for example, Van Reis et al., The Journal of Immunology 133, 758 (1984) who reduced plasma protein levels from leukocyte cell cultures thereby reducing impurities of crude endogenously produced human gamma interferon. (See also Van Reis et al., Methods in Enzymology 119, 77). In November, 1982, at the Third Annual International Congress for Interferon Research in Miami, Florida, Van Reis et al., reported on the use of cross-flow filtration to reduce levels of autologous plasma protein in recovering endogenously-produced human gamma interferon from a human cell culture. However, the amount of interferon recovered could not be increased with further removal during production.
It is an object of the present invention to provide processes for the (large-scale) production and recovery of biologically active human tissue-type plasminogen activator from a culture of human tissue-type plasminogen activator producing host cells. It is a further object of the present invention to provide such processes for production of biologically active human tissue-type plasminogen activator which is substantially free from proteolytic degradation, deglycosylation, inhibition by various protease inhibitors, and contamination and aggregation with serum components.